Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble

Summary. Hepatitis B core antigen is one of the most promising protein carriers of foreign epitopes of various human and animal pathogens. Chimeric HBcAg particles can be used as effective artificial immunogenes. Unfortunately, not all chimeric proteins are able to be particulated. The dependence of correct or incorrect folding of chimeric proteins on physical and chemical properties of inserts was studied with the help of ProAnalyst, SALIX and QSARPro computer programs. We have found that insertion of amino acids with high hydrophobicity, large volume, and high β-strand index prevent self-assembling chimeric proteins. These factors are most important for the C-termini of inserts. Recommendations for obtaining correct folding of chimeric HBcAg particles have been given. Received August 8, 1999, Accepted September 26, 1999     

Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.     

Comparative genetic analysis of the hexaploid wheat Triticum petropavlovskyi Udasz. et Migusch. and Triticum aestivum L

A poorly studied species of hexaploid wheat Triticum petropavlovskyi Udacz. et Migusch. was compared with common wheat Triticum aestivum L. by means of monosomic and genetic analyses of F2 hybrids. Triticum petropavlovskyi was found to carry 13 dominant genes determining its morphological and physiological characters and regular bivalent conjugation of chromosomes. These genes were allelic to the respective genes of common wheat and were located in the same chromosomes. The modes of gene interaction were also the same. There was simple dominance for most genes studied and complementary interaction for the genes of hybrid dwarfism and hybrid necrosis. Triticum petropavlovskyi had the following dominant genes: Hg (downy glume); Rg1 (red glume color); Hl (downy leaf); Hn (downy node); Pa (pubescent auricles); Q (speltlike ears); D1 (grass-clump dwarfism); Ne1 (hybrid necrosis); Ph1 and Ph2 (genes of bivalent conjugation preventing homoeologous chromosomes from pairing); and Vrn1, Vrn2, Vrn3, and Vrn4 (genes of the spring habit). The gene Vrn1, which caused an increase in ear emergence time and a pronounced response to vernalization, was poorly expressed. T. petropavlovskyi was earlier demonstrated to have a species-specific gene P or Eg (elongated glume), which was not allelic to the gene Eg of the tetraploid T. polonicum L. The data obtained indicate that T. petropavlovskyi has originated from T. aestivum via mutations.     

Morphometric and cytogenetic characteristics of haploid tomato plants

Stable differences have been found between haploid and diploid Mikado tomato plants with respect to quantitative characteristics of their vegetative and generative organs, whereas these plants were phenotypically similar with respect to qualitative characteristics, such as the bush habitus and the shapes of leaves, flowers, and fruit. A simple indirect method based on counting the chloroplasts in guard cells has been suggested to diagnose haploids. Analysis of meiosis during microspore formation in haploids has revealed considerable anomalies leading to heterogeneity and a high sterility rate of pollen, which has been confirmed by differences in DNA content. The data obtained suggest that formation of fertile gametes is accounted for by the absence of reduction division. It has been hypothesized that meiosis in haploids induces the genotypic variation that is subject to selection.     

Divergence of karyofunds in sibling species of the plumosus group (Diptera: Chironomidae)

Cytogenetic differentiation of eight sibling species of the plumosus group was examined. The karyofunds of these sibling species were shown to diverge incompletely. In each species karyofund, the banding sequences homologous to those of the remaining species of this group were revealed. The number of banding sequences that displayed interspecific homology varied from 3 to 13 per species karyofund. In a species karyotype, the homologous sequences were localized to chromosome arms 1-6. Both similar and contrasting frequencies of homologous banding sequences were observed in karyofunds of different sibling species. The average cytogenetic distance between sibling species of the plumosus group was 2.618 +/- 0.400. The presence of species-specific banding sequences, the absence of homologous banding sequences in some chromosome arms of the karyotype, and different frequencies of the homologous banding sequences determined the cytogenetic divergence of the sibling species.     

Membrane redistribution of the Escherichia coli MinD protein induced by MinE

Escherichia coli cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown (D. Raskin and P. de Boer, Cell 91:685-694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD.     

Conformation and molecular and ionic transformations of polycytidylic acid immobilized in multilayer Langmuir and polyelectrolyte films

Multilayer films of complexes of polycytidylic acid with dioctadecyldimetylammonium were obtained by the Langmuir-Blodgett method (LB films), and complexes of poly(C) with polycations (poly-L-lysisne, polyethyleneimine, polyallylamine) were obtained by the method of alternate adsorption (polyionic assembly) from solutions of oppositely charged polyelectrolytes on the solid carrier (SA films). It was shown that poly(C) exists in SA films in a single-stranded state irrespective of whether in the starting solution it occurred in the single-stranded nonprotonated or double-stranded protonated conformation. Conversely, in the LB film poly(C) preferred to be in a double protonated conformation. UV-spectra of water-insoluble LB and SA films at different pH values of surrounding water medium were investigated. Proton titration curves of poly(C) immobilized in LB films were obtained. The analysis of the shape of titration curves showed that the molecular-ionic transformation of poly(C) in LB films is accompanied by both the conformational transition of the polynucleotide and the molecular rearrangement in the whole film. Poly(C) was found to transform from the double- to single-stranded state and vice versa in the "deprotonation-protonation" cycle of LB film due to cooperative release/binding of hydrogen ions by cytosine bases. In contrast, poly(C) "protonation-deprotonation" in SA films occurred without conformational transitions of the polynucleotide. As opposed to poly(C) in solution a rather big hysteresis of forward and back titration curves was found for both types of multilayer films, indicating molecular rearrangements in films. The reason for the structural transformations of poly(C) upon fabrication of LB or SA films and the mechanism of molecular ionic transformations of poly(C) in films are discussed in terms of a simple model of ion exchange. An assumption about the nature of structural transformations of LB and SA films during their protonation-deprotonation is put forward.     

The mechanism of the proton transfer: an outline

A brief summary of the principal notions of the quantum-mechanical theory of the charge transfer reactions has been presented. In the framework of this theory, the mechanism of the proton transfer consists in the classical medium reorganization that equalizes the proton energy levels in the initial and final states, and a consequent proton transfer via a quantum-mechanical underbarrier transition. On the basis of this mechanism, factors influencing the proton transfer probability, and hence kinetic isotope effect, have been discussed; among them are the optimum tunneling distance, the involvement of the excited vibrational states, etc. Semi-classical and quantum-mechanical treatments of the Swain-Schaad relations have been compared. Some applications to enzymatic proton-transfer reactions have been described.     

Oxidative regulation of fatty acid-induced tau polymerization

Alzheimer's disease (AD) is characterized by the presence of amyloid-positive senile plaques and tau-positive neurofibrillary tangles. Aside from these two pathological hallmarks, a growing body of evidence indicates that the amount of oxidative alteration of vulnerable molecules such as proteins, DNA, and fatty acids is elevated in the brains of AD patients. It has been hypothesized that the elevated amounts of protein oxidation could lead directly to the formation of neurofibrillary tangles through a cysteine-dependent mechanism. We have tested this hypothesis in an in vitro system in which tau assembly is induced by fatty acids. Using sulfhydryl protective agents and site-directed mutagenesis, we found that cysteine-dependent oxidation of the tau molecule is not required for its polymerization and may even be inhibitory. However, by adjusting the oxidative environment of the polymerization reaction through the addition of a strong antioxidant or through the addition of an oxidizing system consisting of iron, adenosine diphosphate, and ascorbate, we found that oxidation does play a major role in our in vitro paradigm. The results indicated that fatty acid oxidation, the amount of which is found to be elevated in AD patients, can facilitate the polymerization of tau. However, "overoxidation" of the fatty acids can inhibit the process. Therefore, we postulate that specific fatty acid oxidative products could provide a direct link between oxidative stress mechanisms and the formation of neurofibrillary tangles in AD.     

A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis

mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.